The proposed research concerns mechanisms of regulation of two key enzymes in lipid and carbohydrate metabolism of many species: glucose 6-phosphate dehydrogenase (G6PD) and acetyl-CoA carboxylase (AC). Both enzymes undergo large changes in the mammary gland from low levels in pregnancy, to very high levels during lactation, reverting to low levels after the young are weaned. G6PD is an amphibolic enzyme for the facultative anaerobic microorganism Leuconostoc mesenteroides which catalyzes generation of either NADH or NADPH during its heterolactic fermentation of glucose. G6PD in the chemolithotroph Thiobacillus ferrooxidans also utilizes both NAD ion and NADP ion. Comparative studies are proposed on the regulatory mechanisms of these three G6PDs and on their kinetic mechanisms and active-site amino acids. Emphasis in this research is on the relationship between catalytic activity and enzyme conformation and is partly an extension of our previous work which showed that both the mammary and the L. mesenteroides G6PDs exist in two forms, apparently conformational isomers, with different catalytic activities with NAD ion and NADP ion. A new isolation procedure for animal G6PDs is also proposed, based on affinity chromatography, and applicable to rapid isolation of G6PDs in high yield. This procedure is aimed especially at the isolation of G6PDs from human erythrocytes occuring in normal persons and those with various hereditary anemias. Research is also proposed on characterization of an estrogen-responsive G6PD isoenzyme described by Hilf, present in pregnant rat mammary glands and certain mammary cancers, and detailed comparison of this isoenzyme with the one we normally isolate from the lactating mammary gland. Our studies on AC are aimed at ascertaining whether this enzyme is membrane-bound in the mammary gland and, if so, how its activity is regulated under these circumstances. We will attempt fine-structural localization studies and perform detailed investigation of our previously described activation of AC by phospholipids in the absence of citrate.